title: Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: the case of melanocortin 4 receptors.
abstract: We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.
authors.affiliation: University of Tartu, Institute of Chemistry, Ravila 14a, Tartu, Estonia; Competence Centre on Reproductive Medicine & Biology, Tiigi 61b, Tartu, Estonia.
authors.forename: Santa
authors.initials: S
authors.lastname: Veiksina
authors.forename: Sergei
authors.initials: S
authors.lastname: Kopanchuk
authors.forename: Ago
authors.initials: A
authors.lastname: Rinken
mesh_terms: D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D002232:Carbocyanines / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry* / Q000235:genetics; D017476:Receptors, Neuropeptide Y / Q000235:genetics; D061987:Sf9 Cells
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D008024:Ligands; C470072:MC4R protein, human; D044105:Receptor, Melanocortin, Type 4; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor
keywords: Binding kinetics; Budded baculovirus; Fluorescence anisotropy; G protein-coupled receptor; Global analysis; Melanocortin 4 receptor